Tracer reagents that enhance reaction-product analysis

ABSTRACT

A composition suitable for formulation of an enzymatic reaction mixture, the composition comprising a reaction component essential for an ex-vivo non-polymerase enzymatic reaction in which a substrate is catalyzed by an enzyme in a reaction mixture to form a product, and a tracer compatible with the enzyme, the composition being substantially free of the substrate.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/610,935, filed Jul. 6, 2000, which claims priority from U.S.Provisional Patent Application Ser. No. 60/143,009 filed Jul. 9, 1999,the content of each of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention generally relates to reagents that are essentialfor an enzymatic reaction and that enhance reaction-product analysis. Inspecific, preferred embodiments, the invention provides compositions ofessential components which facilitate subsequent chromatographic orelectrophoretic analysis.

Enzymes are frequently used in laboratories to catalyze a variety oftransformations. Typical enzymes which have been utilized includeproteases, peroxidases, oxidases, kinases, amylases, and several nucleicacid modifying enzymes such as DNA polymerases, RNA polymerases,ligases, kinases, restriction endonucleases, phosphodiesterases, DNases,exonucleases, RNases, and phosphatases. The nucleic acid-modifyingenzymes have been frequently used in molecular biology laboratories aspart of procedures such as polymerase chain reaction (“PCR”),sequencing, southern hybridization analysis, restriction endonucleaseanalysis, RNase protection, and the production of labeled probes.

The steps involved in performing an enzyme catalyzed transformation cangenerally be categorized as reaction mixture formulation, enzymaticreaction, reaction product characterization, and reaction product use.The steps of mixture formulation, product characterization, and productuse are labor intensive. The formulation of enzyme reaction mixturesentails combining reaction components which are essential for theenzymatic reaction into a reaction mixture. The reaction mixture is thenincubated under conditions favorable for the enzymatic reaction to takeplace, and for a time sufficient to allow the enzymatic reaction toproceed substantially to completion. The reaction mixture is typicallyanalyzed to evaluate the characteristics of the products formed. Thisanalysis often entails a chromatographic or electrophoretic procedure toseparate and evaluate the reaction products, and to determine whetherthe enzymatic reaction has proceeded to completion. Downstreamapplications entail a wide variety of varied uses for the products ofenzymatic reactions, such as utilization in manufacturing, and furtherprocessing of the product with enzymes or chemical processes. In thecase of molecular biological enzymatic reactions, examples of downstreamapplications are transformation of prokaryotic or eukaryotic cells,detection of complementary sequences by southern or northernhybridization, sequencing, phosphorylation, dephosphorylation, ligation,restriction digestion, endonucleolytic digestion, exonucleolyticdigestion, and purification.

Procedures such as liquid chromatography and polyacrylamide gelelectrophoresis (“PAGE”) have been frequently used to analyze theresults of the enzymatic reactions by separating the reaction productsby, for example, molecular weight. The results of the modification ofnucleic acids by enzymes such as DNA polymerase have typically beenanalyzed by subjecting the reaction products to electrophoresis throughpolyacrylamide or agarose gels.

To analyze enzymatic reaction products using chromatography orelectrophoresis, the sample to be analyzed has often been combined withcomponents which assist the operator in performing the separation. Onesuch component is a “tracer”, which is a detectable moiety such as a dyewhich is generally added to the sample immediately before loading thesample onto the chromatography column or electrophoresis gel. The tracermigrates in the medium in the same direction as the sample to indicatethe progress of the separation.

Another reagent, termed “high density agent” herein, has also beencommonly utilized in electrophoretic analysis of the enzymatic reactionproducts. High density agents are generally water soluble, denseliquids, such as a solution of sucrose or glycerol, which have beenmixed with the sample, usually after the enzymatic reaction is complete,to increase the sample density. The increased density of the sampleresulting from mixing the sample with the high density agent aids, forexample, in loading the sample into a well of an electrophoresis gel byallowing the sample, when pipetted into the top of the well, to “fall”through the less-dense electrophoresis buffer solution to the bottom ofthe well.

High density agent and tracer have been combined with reactionproduct-containing samples to be electrophoretically separated. Thecombination of high density agent and tracer is generally termed“loading buffer”.

While tracers and loading buffers have usually been mixed with thesample after the enzymatic reaction is complete, as diagramed in FIG. 1,their use before the commencement of nucleic acid-modifying enzymaticreactions is also known, as diagramed in FIG. 2. Hoppe et al.,BioTechniques 12:679-680 (1992) describe combining a solution of sucrose(up to 30%) and certain dyes (cresol red, tartrazine, or yellow foodcoloring #5) with an enzyme reaction mixture containing all othercomponents for PCR. After the PCR procedure, the samples were reportedlyloaded directly onto an agarose gel for electrophoretic analysis. Theauthors noted that several dyes and heavy components were inhibitory tothe Taq polymerase enzyme used, but that sucrose, cresol red,tartrazine, and yellow food coloring #5 were compatible with Taq.

Certain commercially available products provide tracer or loading bufferfor use in enzyme reaction mixes for PCR. A thermostable polymerasepreparation, Red Hot DNA Polymerase, is available from AdvancedBiotechnologies and reportedly contains a red dye for use to indicateenzyme addition in the enzyme reaction mixture. There are also twoproducts available which comprise a red tracer and a high density agent,for addition to a PCR reaction mixture before amplification. One, calledRediLoad, is available from Research Genetics, Inc., and the other,called Rapid-Load™, is available from OriGene Technologies. Thesereagents must be added to the reaction mixture in a separate pipettingstep.

SUMMARY OF THE INVENTION

Despite the efforts and improvements made in the prior art,inefficiencies in reaction mixture formulation and reaction productanalysis still exist. In particular, there is no product to date whichcombines an essential component for an enzyme reaction with a tracerand/or high density agent which can be used in an enzymatic reaction andprovide sufficient tracer and/or high density agent such that theproduct of the enzyme reaction could be directly evaluated in achromatographic or electrophoretic procedure without supplyingadditional tracer or high density agent. Such a product (“analysisreagent composition”) would provide additional advantages over theproducts currently available because it would (1) indicate reagentaddition into the enzymatic reaction mix, and (2) eliminate the need forseparately adding a loading buffer since the loading buffer componentsare added along with the essential reagent.

Among the several objects of the invention, therefore, is the provisionof compositions for use in formulating enzymatic reaction mixtures thatoffer improved efficiencies in connection with the labor intensiveprotocols for reaction mixture formulation, and reaction productcharacterization.

The invention is thus generally directed to the provision of acomposition comprising an essential component of an enzymatic reactioncombined with a tracer which is compatible with the enzyme, where thecomposition contains an essential absence of the substrate. Thecomposition can have a density at least about 1.01 g/cm³. Thiscomposition is particularly useful for any enzyme reaction wherepost-reaction processing or analysis is benefited by the tracer and/orincreased density of the reaction mixture. In particular, suchcompositions for polymerase and restriction enzyme reactions areprovided, where the presence of the tracer and/or increased density isuseful for post-reaction electrophoretic analysis. Methods for usingthese compositions, and methods for preparing these compositions arealso provided.

The invention is directed, therefore, to a composition which is suitablefor formulation of an enzymatic reaction mixture, the compositioncomprising a reaction component essential for an ex-vivo non-polymeraseenzymatic reaction in which a substrate is catalyzed by an enzyme in areaction mixture to form a product, and a tracer compatible with theenzyme, wherein the composition is substantially free, or has anessential absence, of the substrate. These compositions can furthercomprise a density of at least about 1.01 g/cm³.

The present invention is also directed toward a composition comprising areaction component essential for an ex-vivo enzymatic reaction in whicha substrate is catalyzed by an enzyme in a reaction mixture to form aproduct and a tracer compatible with the enzyme, the composition beingsubstantially free or having an essential absence of the substrate andhaving an optical density greater than about 5 at a visible wavelengthof maximal tracer absorbance.

The present invention is further directed toward a compositioncomprising a reaction component essential for an ex-vivo polymerasereaction in which a nucleic acid polymer product complementary to anucleic acid polymer template is prepared, and a tracer compatible withthe polymerase, the composition being substantially free or having anessential absence of the template and has an optical density greaterthan about 5 at a visible wavelength of maximal tracer absorbance. Thesecompositions can also comprise a density of at least about 1.01 g/cm³.

The present invention is still further directed toward a composition foran enzymatic reaction component which comprises a reaction componentessential for an ex-vivo enzymatic reaction in which a substrate iscatalyzed by an enzyme in a reaction mixture to form a product, and analkaline earth-metal salt of an anionic tracer.

The present invention is also directed toward a composition whichcomprises a reaction component essential for an ex-vivo enzymaticreaction in which a substrate is catalyzed by an enzyme in a reactionmixture to form a product, and a tracer selected from the groupconsisting of acid red 106, acid red 4, acid red 1, amaranth, and acidviolet 5, or a salt thereof.

The present invention is further directed toward a compositioncomprising a reaction component essential for an ex-vivo enzymaticreaction in which a nucleic acid polymer substrate is enzymaticallycleaved by a restriction enzyme in a reaction mixture to form arestriction product, and a tracer compatible with the restrictionenzyme, wherein the composition contains an essential absence of thesubstrate. These compositions can also comprise a density of at leastabout 1.01 g/cm³.

The present invention is still further directed toward an improvement ina method for a polymerase reaction that comprises forming a reactionmixture comprising a polymerase, a nucleic acid polymer template, atracer compatible with the polymerase, and other components essentialfor the polymerase reaction, creating a nucleic acid polymer productcomplementary to the nucleic acid by enzymatic reaction, analyzing theproduct of the enzymatic reaction by an electrophoretic protocol, andobserving the tracer during the electrophoretic protocol withoutproviding additional tracer beyond that which was included in thereaction mixture. The improvement comprises supplying the tracer to thereaction mixture in a composition that comprises the tracer and theenzyme or another essential component, the composition beingsubstantially free or having an essential absence of the nucleic acidpolymer template. A further improvement is in the reaction mixturehaving a density at least about 1.01 g/cm³.

The present invention is further directed toward an improvement in amethod for a polymerase reaction that comprises forming a reactionmixture comprising a polymerase, a nucleic acid polymer template, atracer compatible with the polymerase, and other components essentialfor the polymerase reaction, creating a nucleic acid polymer productcomplementary to the nucleic acid by enzymatic reaction, analyzing theproduct of the enzymatic reaction by an electrophoretic protocol, andobserving the tracer during the electrophoretic protocol. Theimprovement comprises supplying the tracer to the reaction mixture in acomposition that comprises the tracer and the enzyme or anotheressential component, the composition being substantially free or havingan essential absence of the nucleic acid polymer template, wherein thetracer supplied to the reaction mixture is of adequate character andsufficient quantity to be visible during the electrophoretic protocol.

The present invention is also directed toward a method for a restrictionenzyme reaction, the method comprising forming a reaction mixturecomprising a restriction enzyme, a nucleic acid polymer substrate, atracer compatible with the restriction enzyme, and other componentsessential for the enzymatic reaction, enzymatically cleaving the nucleicacid polymer substrate to form a restriction product, analyzing theproduct of the cleavage reaction by an electrophoretic protocol, andobserving the tracer during the electrophoretic protocol withoutproviding additional tracer beyond that which was included in thereaction mixture. The density of the reaction mixture can also be atleast about 0.01 g/cm greater than the liquid phase utilized in thechromatographic or electrophoretic protocol.

The present invention is also directed toward a method for a restrictionenzyme reaction, the method comprising forming a reaction mixturecomprising a restriction enzyme, a nucleic acid polymer substrate, atracer compatible with the restriction enzyme, and other componentsessential for the enzymatic reaction, enzymatically cleaving the nucleicacid polymer substrate to form a restriction product, analyzing theproduct of the cleavage reaction by an electrophoretic protocol, whereinthe tracer supplied to the reaction mixture is of adequate character andsufficient quantity to be visible during the electrophoretic protocol.

The present invention is further directed toward a method for forming anenzymatic composition, the method comprising combining a reactioncomponent with a tracer, the reaction component being essential for anenzymatic reaction in which a substrate is catalyzed by an enzyme in areaction mixture to form a product, the tracer being compatible with theenzyme, and the resulting composition having an optical density greaterthan about 15 at a visible wavelength of maximal tracer absorbance. Aliquid which is compatible with the enzyme can also be added, whereinthe liquid increases the density of the composition to at least about1.1 g/cm³.

The present invention is still further directed toward a method forforming an enzymatic composition, the method comprising combining areaction component with a tracer, the reaction component being essentialfor a polymerase reaction in which a nucleic acid product is polymerizedfrom a complementary nucleic acid template, the tracer being compatiblewith the enzyme, and the resulting composition having an optical densitygreater than about 5 at a visible wavelength of maximal tracerabsorbance. A liquid which is compatible with the enzyme can also beadded, wherein the liquid increases the density of the composition to atleast about 1.1 g/cm³.

Other features, objects and advantages of the present invention will bein part apparent to those skilled in the art and in part pointed outhereinafter. All references cited in the instant specification areincorporated by reference. Moreover, as the patent and non-patentliterature relating to the subject matter disclosed and/or claimedherein is substantial, many relevant references are available to askilled artisan that will provide further instruction with respect tosuch subject matter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides compositions comprising an essentialcomponent to an enzyme reaction and a tracer which can allowpost-reaction analysis without further tracer addition. Thesecompositions (“analysis reagent compositions”) can also comprise a highdensity agent which can eliminate the need for further addition of ahigh density agent during post-reaction analysis. Methods of using thesecompositions, and methods of preparing them are also provided.

Prior art methods of using loading buffer components always added thesecomponents to the reaction mixture, either before or after the enzymaticreactions are executed. Such methods require an extra step to add theloading buffer components. An improvement over the prior art in thepresent invention is the provision of analysis reagent compositionscomprising an essential component for an enzymatic reaction combinedwith the loading buffer components. As diagrammed in FIG. 3, theapproach of the present invention offers further efficiencies that werenot achieved with prior art protocols. The loading buffer components arepassively added with the essential component, thus eliminating the extraaddition step required in the prior art to add the loading buffer. Inlaboratories where many enzyme reaction mixtures are prepared, thecompositions and methods of the present invention can thus eliminate aconsiderable amount of work. These analysis reagent compositions areparticularly useful for molecular biological enzyme reactions,particularly polymerase reactions and restriction enzyme reactions,since these reactions are often repetitively performed understandardized conditions.

The procedures disclosed herein which involve the molecular manipulationof nucleic acids are known to those skilled in the art. See generallyJoseph Sambrook et al. (1989), “Molecular Cloning, A Laboratory Manual”,second ed., Cold Spring Harbor Laboratory Press.

As used herein, the term “substrate” encompasses a component of anenzymatic reaction mixture which is a reactant in the reaction catalyzedby the enzyme. For example, in a restriction enzyme reaction, thesubstrate is the nucleic acid polymer which is cut by the enzyme. In apolymerase chain reaction, the substrates include the primer which isextended by the enzyme, and the nucleotides which are added to thegrowing nucleic acid polymer.

The present invention provides compositions comprising a reagent whichis essential for an enzymatic reaction (“essential reagent”) and atracer which is compatible with the enzyme. A high density agent mayalso be included in these compositions. Methods are also providedwherein the compositions are used in an enzymatic reaction and theresults are subsequently analyzed by electrophoresis or chromatographyin a water soluble solvent.

The compositions and methods of the present invention are useful inconjunction with any enzymatic reaction where the reaction products aresubsequently analyzed by a chromatographic or electrophoretic method.Enzymes which can be employed include those which modify or degradeproteins, lipids, carbohydrates, and metabolites, such as any kinase,protease, lipase, amylase, peroxidase, oxidase, oxygenase, anddehydrogenase. Enzymes which modify, cut, or synthesize nucleic acidsare particularly suitable to be used with the present invention.Examples include any ligase, phosphodiesterase, DNase, exonuclease,RNase, phosphatase, kinase, terminal transferase, reverse transcriptase,restriction endonuclease, RNA polymerase, and DNA polymerase. Enzymeswhich are preferred for use with this invention are restrictionendonucleases and DNA polymerases. More preferred are DNA polymerases;even more preferred are any thermostable DNA polymerase; most preferredis wild-type or modified Taq polymerase. These enzymes can be in anyconcentration which is useful for performing an enzymatic reaction.Preferred concentrations of Taq polymerase in the compositions of thepresent invention are 0.033-10 units/μl, more preferred concentrationsare 0.06-5 units/μl, the most preferred concentration is 1 unit/μl.Preferred concentrations of restriction endonucleases in thecompositions of the present invention are 0.1-1000 units/μl, morepreferred concentrations are 1-100 units/μl, most preferredconcentrations are 5-40 units/μl.

Tracers which can be used in this invention include detectable compoundswhich can be incorporated into the reaction mixture and not interferesignificantly with the enzyme reaction. Such a tracer is designatedherein as “compatible” with the enzyme. It is preferred that thiscompatibility be such that an enzyme composition with the tracer has atleast 95% of the activity of the same composition without the tracer.More preferably, the tracer-enzyme composition has at least about 97%activity of the composition without the tracer, even more preferably atleast about 99% activity, and most preferably about 100% activity. Thetracer should also be stable enough in the tracer-enzyme composition toretain its compatibility with the enzyme even after a long storageperiod at an appropriate temperature, e.g. 1 year or more at −20° C.

The detectable signal imparted by the tracer can be visual, such as thatimparted by a dye or fluorescent compound. The tracer can also impart aradioactive, electrochemical, spectrophotometric, or any other type ofsignal which can be detected sensually or with an instrument and whichcan serve as a useful marker in an analysis subsequent to the enzymereaction. Preferred are tracers which impart a visual signal. The mostpreferred tracers are dyes which are colored under the conditions thatthe analysis is performed. Any color dye which is visible during thepost-reaction analysis can be used; preferred are dyes which have a peakvisible absorbance wavelength at between 430 and 617 nm; most preferabledyes have a peak visible absorbance wavelength at between 500 and 535nm.

While any tracer compatible with the enzyme can be useful in the presentinvention, preferred tracers are highly soluble in the liquid phase ofthe post-reaction chromatographic or electrophoretic procedure. Thetracer is preferably an anionic tracer. Particularly preferred tracersare anionic tracers such as salts of organic acid dyes or sulfonic aciddyes. The preferred salt counterion is an earth metal, most preferablyCa⁺⁺ or Mg⁺⁺. Where the post-reaction analysis is an electrophoresis ofa nucleic acid, preferred tracers are anionic dyes. Preferredconcentrations of the tracer in an analysis reagent composition areconcentrations for which the composition has an optical density (OD) ofbetween about 5 and about 500; most preferred is about 300. In enzymereaction mixtures prepared from the analysis tracer composition thepreferred tracer concentration has an OD of between 1 and 100; morepreferred is 15 to 50; most preferred is 15.

Commercial preparations of tracers are often inhibitory to enzymeactivity when used in the concentrations recited above. This inhibitioncan often be overcome by further purification of the tracer, for exampleby reverse phase desalting, recrystalization, acid precipitation, orchromatographic methods such as reverse phase, normal phase, or ionexchange chromatography. Where the tracer is an anion, such as disclosedin Example 1 below, enzyme inhibition can also be overcome by replacingthe counterion with an alkaline earth-metal. Preferred alkalineearth-metals for this purpose are Ca⁺⁺ and Mg⁺⁺; most preferred is Mg⁺⁺.

High density agents useful for the present invention include any solutein which the tracer is soluble and which is compatible with the enzymewhen diluted in the final reaction mixture, and which is dense enough toassist in the addition of reaction mixture to the analytical process. Toprovide such assistance, the density of the reaction mixture should beat least about 0.01 g/cm³ greater than the density of the liquid phaseof the analytical process (e.g. the electrophoresis or chromatographicbuffer). A somewhat higher density (for example, about 0.05 g/cm³greater than the analytical liquid phase) would provide greaterassistance in the addition of the reaction mixture, and is thus morepreferred. These densities may be provided using the preferred densityof an essential enzyme component/tracer/high density agent compositionof about 1.14 g/cm³. Higher densities are also useful, however, providedthey are. compatible with the enzyme at the concentration used in theenzyme reaction.

Examples of solutes which are generally compatible with enzymes at theconcentrations required to provide sufficient density are sucrose orother sugars, glycerol, and betaine (trimethylglycine). Glycerol ispreferred. Glycerol at a concentration of 1.5% in water is about 0.01g/cm³ more dense than water, and would thus provide assistance inapplying a sample to an analytical process where water is the liquidphase. Glycerol at a concentration of 50% in water has a density ofabout 1.14 g/cm³. Thus, 50% glycerol is preferred as the high densityagent in an essential enzyme component/tracer/ high density agentcomposition.

A scheme which is useful for evaluating the compatibility andeffectiveness of a tracer and/or high density agent (“loading buffercomponent”) for this invention is shown in FIG. 4. In this scheme, thedesired physical characteristics of the loading buffer component isdetermined. For example, the desired color and charge of a dye to beused as a tracer is decided. Next, a set of candidates (e.g. red,anionic dyes) is assembled for testing to evaluate other desiredproperties (e.g. enzyme compatibility, lack of interference in genetictransformation protocols, etc). The candidates are then tested for thesedesired properties, preferably by performing the least laborious testsfirst, in order for the largest amount of undesirable candidates to beeliminated by the least amount of screening. When desirable loadingbuffer components are selected, the formulation of loading buffercomponent and essential reagent is prepared, and its effect on theenzyme reaction and subsequent analysis is characterized.

Any essential reagent can be selected to be combined with the loadingbuffer component to formulate a composition of the present invention.The selection of an essential reagent for this purpose can depend onfactors such as:

-   -   whether it is desired to be able to determine if the essential        reagent has been added to the enzyme reaction mixture. For        example, if the essential reagent is an enzyme and the loading        buffer component is a dye, then one can easily determine if the        enzyme has been added to the reaction mixture by determining if        the reaction mixture is colored.    -   whether the essential reagent might be added at varying        concentrations in several reaction mixtures, or whether there        are alternative formulations of essential reagents which might        be added. For example, a particular buffer solution can be used        at different concentrations by several different restriction        endonucleases. See Joseph Sambrook et al. (1989), “Molecular        Cloning, A Laboratory Manual”, second ed., Cold Spring Harbor        Laboratory Press, at pp. 5.28-5.31. The concentrated buffer        solution may not be a preferred essential reagent to combine        with the loading buffer components under those conditions, since        the final reaction mixtures could have varying concentrations of        the loading buffer components, depending on the enzyme used.        However, where the concentrated buffer solution is generally        always diluted a particular amount, such as with 10× buffers        which are often provided for particular enzymes (e.g.        restriction enzymes), the loading buffer components can be        usefully provided in combination with these solutions.

Examples of essential reagents which can be combined with loading buffercomponents to formulate a composition of the present invention are:enzyme, concentrated enzyme buffer (e.g. 10× buffer), a nucleotide orprimer reagent in the case of DNA or RNA polymerases, or a coenzyme suchas NADPH or ATP. The preferred essential agent for this purpose is theenzyme, since it is often desirable to be able to ascertain if enzymeaddition has taken place, and since enzyme concentrations in reactionmixtures are generally not widely varied. A colored enzyme formulationalso has the advantage of allowing one to determine if complete mixingof the enzyme has taken place. If the solution is uniformly colored thenthe enzyme is uniformly distributed. Also, since a colored formulationis more readily visible than a clear formulation, a colored enzymeformulation also facilitates pipetting of the small volumes of enzymewhich are often added to enzymatic reaction mixtures.

Inclusion of loading buffer components with the substrate is usually notpreferred because the substrate composition and concentration oftenvaries between individual enzyme reactions. For a PCR reaction, however,the loading buffer components can be advantageously added with thenucleotide substrates, since the concentration of these reagentsgenerally do not vary between individual PCR reactions.

As contemplated by the present invention, the analysis of the product ofan enzyme reaction can be by any method which is suitable for theproduct in question. Chromatographic and electrophoretic methods areparticularly suitable. Suitable chromatographic methods include liquidchromatography (“LC”), particularly gel permeation chromatography. InLC, a high density agent would facilitate the loading of the reactionmixture containing the product onto a chromatographic column, and avisible tracer would allow one to follow the progression of the samplethrough the column.

For applications where the product to be analyzed is a nucleic acidpolymer, electrophoretic methods are preferred. In this regard, thecompositions and methods of the present invention are useful for agarosegel electrophoresis (e.g. to analyze products of PCR, restrictionendonuclease digestion, ligation reactions, etc), and polyacrylamide gelelectrophoresis (e.g. analysis of sequencing reactions). Polyacrylamidegel electrophoresis is also facilitated by the present invention whenused to analyze protein products of enzymatic reactions.

The following examples illustrate the invention, but are not to be takenas limiting the various aspects of the invention so illustrated.

EXAMPLE 1 Identification and Formulation of a Taq DNA Polymerase withTracers and High Density Reagent

A composition conforming to the present invention was developed tofacilitate analysis of products resulting from PCR. The compositioncomprises an essential PCR component, Taq DNA polymerase, withsufficient glycerol to facilitate the application of the reactionproduct to an agarose gel for electrophoretic analysis. The compositionalso comprises a red dye that aids in visualization and mixing of theenzyme in the reaction mixture. The red dye also serves as a tracer tofollow the progressive movement of PCR products through an agarose gelduring electrophoresis. The color red was selected for aesthetic reasonsand confers no particular advantage as a tracer.

FIG. 5 summarizes the steps taken to develop this composition. Sincenucleic acid products of PCR are highly anionic, they are applied to theagarose gel near the anode and move toward the cathode as theelectrophoresis progresses. Therefore, to be useful as a tracer inelectrophoresis the dye molecule is preferably anionic. FIG. 6summarizes the selection process. From 180+ red dyes (absorbance maxbetween 450 and 570 nm) (Table 1) approximately 40 anionic dyes wereselected (Table 2). TABLE 1 Dyes initially considered Dye λ_(max)Bis-N-methylacridinium nitrate 430 4-(p-Nitrophenylazo)-resorcinol 432Auramine O 432 Martius Yellow 4323′,3″,5′,5″-Tetraiodophenolsulfonephthalein 4336′-Butoxy-2,6-diamino-3,3′-azodipyridine 435 Quinoline Yellow A, spiritsoluble 435 m-Cresol Purple, sodium salt 436 Methyl Red, sodium salt 437Methylthymol Blue, water soluble 438 a-Naphthyl Red 439 Palatine FastYellow BLN 440 Twort Stain 440 Pyrocatechol Violet 441 Acridine Yellow G442 Mordant Brown 33 4422-(5-Bromo-2-pyridylazo)-5-(dimethylamino)phenol 443 Disperse Orange 3443 Acid Yellow 99 445 Thymolphthalein monophosphoric acid, disodiumsalt hydrate 445 Acid Orange 51 446 Eriochrome Cyanine R 446 MalachiteGreen Carbinol base 446 Ethyl Red 447 Chrysoidin 449 Orange G 475 SudanI 476 trans-p-Carotene 478 Fast Yellow 480 Pyrogallol Red 480 DirectBlack 22 481 Crocein Orange G 482 Rosolic Acid 482 Disperse Orange 1 483Eriochrome Red B 483 Orange 11 483 Thorin I 483 Purpurin 485 Quinizarin485 Mordant Brown 1 487 Acridine Orange 488 Para Red 488 Acridine Orange489 Acid Orange 8 490 Astrazon Orange G 490 Fluorescein diacetate 490Fluorescein isothiocyanate, isomer I 490 Quinalizarin 490 Tropaeolin 0490 Zincon 490 Zincon, monosodium salt 490 Fluorescein, water soluble491 Acridine Orange hydrochloride 492 Mordant Brown 48 492 Methyl Redhydrochloride 493 Sudan 11 493 Acid Red 183 494 Reactive Orange 16 494Carminic acid 495 Disperse Red 19 495 Fluoresceinamine, isomer 11 495Fluorescein 496 Fluoresceinamine, isomer I 496 BrilliantYellow 497 CongoRed 497 Acid Red 97 498 Cochineal 498 Arsenazo I 499 Fluorexon 499Benzopurpurin 4B 500 Mordant Brown 4 500 Reactive Red 8 500 AcidAlizarin Violet N 501 Rhodamine 123 dehydrate 501 Darrow Red 502Disperse Red 1 503 Xylidine Ponceau 3RS 503 Acid Red 106 505 Acid Red 88505 Biebrich Scarlet, water soluble 505 Nuclear Fast Red 505 Acid Red 4506 New Coccine 5062-(4-Sulfophenylazo)-1,8-dihydroxy-3,6-naphthalene-disulfonic 507 DirectRed 23 507 Merbromin 507 Methyl Orange 507 Sudan III 507 Toluidine Red507 Acid Red 4 508 Acid Red 8 508 Direct Red 81 5082′,7′-Dichlorofluorescein 509 Brilliant Crocein MOO 510 Chromotrope 2R510 Basic Red 29 511 Acid Red 151 512 Chromoxane Cyanine R 512Quinalizarin 512 Acid Red 37 513 Acid Red 114 514 Chromotrope 2B 514Eosin B 514 Eosin Y 514 Ponceau SS 514 Acid Red 150 515 Chromotrope FB515 Acid Red 40 516 Azocarmine B 516 Mordant Blue 9 516 Reactive Red 4516 Cibacron Brilliant Red 3B-A 517 Disperse Red 13 517 Eosin Bluishblend 517 4,5,6,7-Tetrachlorofluorescein 518 Bordeaux R 518 Oil Red 0518 Acid Violet 7 520 Methyl eosin 520 Ponceau S 520 Rose Bengal,bis(triethylammonium) salt 520 Sudan IV 520 Amaranth 521 Emodin 521Eosin Y, free acid 521 Giemsa Stain 521 Oil Red EGN 521 Purpuri'n 521Azure A eosinate 522 Diiodofluorescein 522 Direct Red 75 522 Eosin B,spirit soluble 522 Jenner Stain 522 Leishman Stain 522 May-GrbnwaldStain 522 Wright Stain 522 Wright Stain, solution in methanol 522 AzureB eosinate 523 Zincon, monosodium salt 523 Acid Blue 120 524 Azure 11eosinate 524 Eosin Y lactone 524 Rhodamine 6G 524 Tetrachrome Stain(MacNeal) 524 Erythrosin B 525 Erythrosin Yellowish blend 525 Ethidiumbromide 525 Acid Violet 5 527 Plasmocorinth B 527 Eriochrome Blue Black2B 528 Quinaldine Red 528 Rhodamine 6G Perchlorate 528 Rhodamine 6Gtetrafluoroborate 528 Sulforhodamine G 529 Violamine R 529 Chromotrope2R 530 Safranine 0 (Y, T) 530 Alum Carmine 531 Carmine 531 Acid Red 1532 Acid Red 106 532 Ethyl Eosin 532 Arsenazo 111, sodium salt hydrate533 Erythrosin B, spirit soluble 533 Sudan Red 7B 533 Ruthenium Red 534Nuclear Fast Red 535 Acid Red 40 538 Alizarin Violet 3R 540 Neutral Red540 Aluminon 542 Rhodamine B 543 Basic Fuchsin 544 Basic Fuchsin,special for flagella 544 Pararosaniline base 544 Rhodamine B base 544Acid Fuchsin, calcium salt 545 Acid Violet 17 545 Aurintricarboxylicacid 545 Aurintricarboxylic acid, trisodium salt 545 Pararosanilineacetate 545 Acid Fuchsin, sodium salt 546 Carbol Fuchsin 547 AlizarinBlue Black B 548 Phloxine B 548 Pyronin Y 548 Rose Bengal 548 BasicFuchsin, biological stain 549 Direct Violet 51 5499-Phenyl-2,3,7-trihydroxy-6-fluorone 552 Bromopyrogallol Red 552Phenolphthalein 552 Rhodanile Blue 552 New Fuchsin 553 Nile Red 553Pyronin B 553 Sulforhodamine B 554 Alizarin Red S monohydrate 556Methylene Violet 3RAX 557 PhenolRed 557 Rose Bengal,bis(triethylammonium) salt 559 Arsenazo III 560 Pinacyanol chloride 560Acid Blue 161 563 Carmine 563 Nigrosin, alcohol soluble 565 Acid Blue113 566 o-Cresolphthalein 566 Alizarin 567 Sulfonazo 111, tetrasodiumsalt 567 Palatine Chrome Black 6BN 569 Brilliant Black BN 570 Cresol Red570 Bromocresol Green (also broad absorbance at 417 nm) 617

TABLE 2 Properties of anionic dyes λ_(max) No Dye (nm) Color EtOH QiagenPCR 1 Orange G 475 X 2 Acid Red 150 515 X 3 Acid Red 88 505 solubility 4Acid Red 106 532 X 5 m-Cresol Purple, sodium salt 436 X 62-(4-Sulfophenylazo)-1,8- 507 X dihydroxy-3, 6-naphthalene- disulfonicacid, trisodium salt 7 Mordant Blue 9 516 X 8 Chromotrope 2R 510 X 9Pyrogallol Red 480 X 10 Reactive Red 4 516 X 11 Disperse Orange 1 483 X12 Congo Red 497 X 13 Direct Red 81 508 X 14 Phloxine B 548 X 15Eriochrome Cyanine R 446 X 16 Acid Violet 17 545 X 17 Chromotrope 2B 514X 18 Zincon, monosodium salt 490 X 19 Methyl Red, sodium salt 437 X 20Acid Orange 8 490 X 21 Rosolic Acid 482 solubility 22 Eosin Y 514 X 23Bordeaux R 518 X 24 Acid Red 106 505 25 Acid Red 4 506 26 Acid Red 1 53227 Bromocresol Green (also broad 617 X absorbance at 417 nm) 28 PonceauS 520 X 29 Benzopurpurin 4B 500 solubility 30 Acid Orange 51 446 X 31Amaranth 521 32 4-(p-Nitrophenylazoo- 432 X resorcinol 33 BiebrichScarlet, water 505 X soluble 34 Martius Yellow 432 X 35 Reactive Orange16 494 X 36 Direct Violet 51 549 X 37 Chromotrope FB 515 X 38 Direct Red75 522 X 39 Acid Violet 5 527 40 Acid Red 97 498 X

Each of these dyes were dissolved in water and those that were notparticularly red (i.e. were too yellow/orange or purple), or lackedsufficient solubility, were removed from consideration (Table 2, thosemarked under “Color”).

The 20 dyes which remained were assayed for their ability to be removedfrom DNA by ethanol precipitation. To 1 μg of lambda DNA enough dye wasadded to yield a highly colored solution. Addition of two volumes of 3Mammonium acetate, then 6 volumes of ethanol followed. The DNA waspelleted by centrifugation. The appearance of a colored pellet causedthe dye to be removed from consideration (FIG. 6, EtOH ppt.; Table 2,those marked under “EtOH”). In the presence of the remaining dyes, oneμg quantities of DNA were purified by solid phase extraction on QiagenPCR product purification columns (Qiagen, Hilden, Germany) according tothe manufacturers protocol. Dyes that yielded colored eluants weredropped from consideration (FIG. 6, labeled “Qiagen”, Table 2, thosemarked under column labeled “Qiagen”). The assays performed to thispoint were done at the onset of the screening because they were theleast laborious.

The dyes that survived these preceding tests were included in a PCRtoxicity study. The dyes were added to PCR reactions at concentrationsconsidered adequate for product performance. Enough of a 2× PCR mastermix consisting of Taq DNA polymerase, 0.1 u/μl (Sigma Chemical Co., St.Louis, Mo.), PCR buffer (2×) (Sigma), dNTP's (200 μM each) (Sigma),α³²PdCTP (Amersham USA, Piscataway, N.J.), target DNA (lambda, 2 ng/μl)(Sigma) and primers (Perkin-Elmer 500 bp control, 2 μM) (Perkin-Elmer,Norwalk, Conn.) was prepared to accommodate all 11 remaining dyes plusthree no dye controls. Ten μl of the 2×0 master mix was dispensed intoreaction tubes followed by addition of 10 μl of aqueous darkly coloreddye solutions or water (controls). The PCR cycling protocol was 20cycles of 94/55/72° C. at one minute each. 20 μl of quench solution (50μg/μl calf thymus DNA, 20 mM EDTA) was added followed by precipitationwith 40 μl 40% trichloroacetic acid (TCA)/4% sodium pyrophosphate(NaPPi). The reactions were filtered on glass fiber filters, washed with5% TCA/2% NaPPi and counted by scintillation methods. Thisquench/precipitation procedure will henceforth be referred to as “TCAprecipitation”. As shown in FIG. 7, some dyes significantly inhibitedthe PCR reaction such that little or no product resulted (i.e. numbers8, 9, 10, 35 and 40). However, other dyes were relatively inert (i.e.23, 24, 25, 26, 31, 39). The dyes that inhibited PCR were dropped fromconsideration (FIG. 6, PCR Tox, Table 2, those marked under “PCR”).Number 23 was dropped because its per cycle yield was substantiallylower than the other dyes (FIG. 7). Per cycle yield was calculatedassuming the overall yield was the per cycle yield raised to the 20^(th)power (FIG. 7).

The dyes which remained after the PCR toxicity screen were furtherscreened for their toxicity on ligation and transformation, twodownstream procedures often carried out using unpurified PCR products.As shown in FIG. 8, ligation in the presence of the remaining dyes (24,25, 26, 31 and 39 lanes 3-7 respectively) was equivalent to the no dyecontrol (lane 2). Lane 1 is a control which had no ligase. Ligationswere carried out as described in the figure legend. The same dyes werealso tested for suitability in a ligation/transformation protocol.Transformation efficiency was not compromised by the presence of dye(FIG. 9). Increased efficiencies, as evidenced in the figure, were notinvestigated. The experiment was performed as summarized in the figurelegend.

On the basis of performance to this point, the remaining dyes wereconsidered substantially equivalent as tracer candidates. Acid violet 5(number 39, Table 2) was chosen as a possible finalist candidate. Usingthis dye in a reaction mix at an absorbance of 10 (at its absorbancemaximum of 527 nm) a PCR reaction was performed. The inclusion of thisdye in a reaction mix resulted in a minimum of 50% loss (relative to thesame reaction without the dye) in PCR product yield (FIG. 10) asmeasured by TCA precipitation. This was considered unacceptable, but wasovercome by further development efforts.

In general, the available dyes, as well as those investigated, arederived from unrelated applications. That is, many are used as fabricdyes, food colorings, biological stains etc. As such, they are availablein various states of purity. In the case of the finalists, they all hadpurities of 80% or less, as determined by the product labels. In furtherexperiments, PCR yields (determined by TCA precipitation) in reactionscontaining various concentrations of acid violet 5 (determined by A₅₂₇)were compared before and after purification of the crude dye by reversephase desalting. PCR product yield was much less susceptible to dyeconcentration for the desalted compound relative to the crude compound(FIG. 11). Since reversed phase desalting is not an attractive methodfor large scale dye purification, acid precipitation followed byammonium hydroxide dissolution/evaporation to produce the ammonium dyewas investigated. This procedure should result in an essentially saltfree product. These procedures (reversed phase desalting andacid/ammonium hydroxide) were carried out for acid red 1 (No. 26, Table2) and acid violet 5 (No. 39). It was found that the ammonium dyes weremore PCR toxic than the sodium (i.e. desalted) dyes (FIG. 12 a). Thedyes were converted to their magnesium salts to further characterize theeffect of counterion identity. This was accomplished by addition ofmagnesium chloride (excess) to a solution of the crude dye. Themagnesium dye that immediately precipitated was recrystallized fromwater. As shown in FIG. 12 a, the Mg salts of both acid red 1 and acidviolet 5 was much less toxic to PCR than either the sodium or ammoniumsalts. In analogy with the magnesium salts, the calcium and zinc saltsof acid red 1 were prepared to investigate whether the effect wasdivalent vs. monovalent cation or cation identity specific. FIG. 12 bdemonstrates that the magnesium salt was least toxic. From these data itwas concluded that the dye was likely sequestering magnesium from thePCR reaction which caused decreased product yields.

A dye formulation of the magnesium salts of 80% acid red 1/20% acidviolet 5 was created (percentages based on absorbance at the wavelengthof each dye's maximum absorbance [acid red 1=531 nm, acid violet 5=528nm]). That particular ratio was used for aesthetic reasons, however anyratio, or either of the dyes individually would be similarly effective.To further investigate the effects of Mg⁺⁺, since the dye is supplyingmagnesium to a magnesium dependent reaction (i.e. PCR), free Mg⁺²concentration contributed by the dye to the reaction was determined.This was determined by varying the magnesium concentration in dyecontaining vs. dye free reactions in a dose response manner. FIG. 13shows that for products ranging from 500 to 3000 bp, the differencebetween red and white Taq at the midpoint of the magnesium concentrationtitrations is approximately 0.4 mM (0.37±0.04). The 10× buffer usuallysupplied with Taq was reformulated to account for this perturbation(i.e. the concentration of MgCl₂ was changed from 15 to 11 mM in the 10×buffer).

A preferred composition was formulated at 1 u/μl Taq polymerase in Taqstorage buffer (consisting of 20 mM Tris-HCl, pH 8.0, 100 mM KCl, 0.1 mMEDTA, 1 mM DTT, 0.5% Tween 20, 0.5% Igepal® CA-630, 50% glycerol inwater) with the magnesium formulation of dye at a total absorbance of300. The dye composition was 80% acid red 1, 20% acid violet 5(100%=absorbance of acid red 1 at λ_(max)+absorbance of acid violet 5 atλ_(max), absorbance of acid red 1=240, acid violet 5=60). Thisformulation is designated “REDTaq™”. When added to a PCR reactionmixture at 0.05 u/μl Taq, the total dye absorbance is 15. The dyecombination at this concentration was visible in a subsequent agarosegel electrophoresis of the completed reaction mix, yet the combinationwas relatively non-toxic to PCR. A lower concentration of the dye in thereaction mixture would be difficult to see during a subsequent agarosegel electrophoresis. As a comparison, the previously discussed prior artTaq-dye formulation, Red Hot DNA Polymerase, has an absorbance of 3.3 at572 nm, and 4.6 at 435 nm. At the recommended concentration in a PCRreaction mixture, Red Hot DNA Polymerase has an absorbance of 0.033 and0.046, at 572 and 435 nm, respectively. Therefore, in contrast toREDTaq™, the Red Hot DNA Polymerase formulation would not be useful as atracer in an electrophoretic analysis of a PCR reaction.

PCR products prepared using the REDTaq™ formulation with the 10× bufferdescribed above (with 11 mM MgCl₂) were compared with conventionalTaq/10× buffer (with 15 mM MgCl₂). FIG. 14 shows a 1% agarose gel ofamplification products resulting from this comparison. From the gel itis apparent that the amplifications using REDTaq™ were equivalent tothose using Taq without dye. The exception to this is amplification ofthe 3 kb fragment, where the amplification with conventional Taq failedfor unknown reasons (Lane 4). However, when product yields were comparedfor a variety of target sizes (FIG. 15), both conventional Taq andREDTaq™ did effectively amplify a 3 kb target. In that comparison,product yield was not compromised by REDTaq™. When a similar comparisonwas made with RediLoad, a commercial formulation of a red loading buffer(without an essential reaction component) which is added before a PCRreaction, the Rediload product reduced PCR product yield byapproximately 10% (FIG. 16), relative to the same reaction withoutRediload.

EXAMPLE 2 Determination of the Compatibility of a Dye with RestrictionEndonucleases

The effectiveness of restriction enzymes in cutting target DNA when adye is present in the reaction mixture was evaluated. A variety ofrestriction enzymes were assayed for the detrimental effect of addingAmaranth (No. 31, Table 2) to a restriction digest as assayed by agarosegel electrophoresis. Nde I-cut pUC19 plasmid was prepared. Thislinearized plasmid was then digested with one of several restrictionenzymes which normally cut pUC19 at a polylinker site. Thus, each enzymewould be expected to yield a product of similar sizes (ranging from 212to 263 bp). The restriction enzyme digests were performed in thepresence, or absence, of Amaranth dye, and at various concentrations ofthe enzyme. The results of this experiment are shown in FIG. 17. Column1 (the leftmost column) contains 100 bp molecular weight ladders.Columns 2-4 contains cleavage products from sequential 5-fold dilutionsof the restriction enzymes in the presence of dye. Columns 5-7 are ascolumns 2-4 but without dye. Columns 2 and 5 contained the restrictionenzyme at {fraction (1/10)}^(th) the suppliers concentration (i.e. theenzyme was considered a 10× concentration). The buffers used for thedigests were as recommended by the supplier. The enzymes used are listedalong the right side of the gel. The relative susceptibility of theenzyme to the dyes' presence is reported next to the enzyme. The sizesof the small bands in the electrophoresis runs range from 212 to 263 bp.The larger, brighter bands contain the full length NdeI-cut pUC19 (2686bp) and/or the larger fragment of the NdeI-cut pUC19 which was also cutwith the test enzyme (2423-2474 bp). +++ equals inert, blank iscompletely toxic. This preliminary experiment reveals that therestriction enzymes tested were relatively insensitive to dye addition.That is, with the exception of KpnI, Amaranth was relatively inert inthese reactions. This experiment was conducted using commerciallyprepared (crude) dye (dye content approximately 90%). Based on thepreviously discussed results with Taq polymerase, a dye screening andpossible cleanup/counterion exchange, similar to that used in Example 1,should result in the discovery of a dye system that would work for amajority of restriction enzymes.

To enable direct gel loading, the restriction digest would have tocontain a component that made the solution more dense than theelectrophoresis buffer. Like the PCR product, glycerol or another highdensity agent should suffice. Generally, star activity (alternate sitecutting of DNA due to high glycerol content) can occur at glycerolconcentrations above 5%. For REDTaq™, solutions as low as 1.5% glycerolwere effective as a high density agent. Thus, it is clearly possiblethat restriction digest reagents could be formulated to contain enoughglycerol or other density increasing solutes to allow for direct gelloading.

Other features, objects and advantages of the present invention will beapparent to those skilled in the art. The explanations and illustrationspresented herein are intended to acquaint others skilled in the art withthe invention, its principles, and its practical application. Thoseskilled in the art may adapt and apply the invention in its numerousforms, as may be best suited to the requirements of a particular use.Accordingly, the specific embodiments of the present invention as setforth are not intended as being exhaustive or limiting of the invention.

1. A method for a restriction enzyme reaction, the method comprisingforming a reaction mixture comprising a restriction enzyme, a nucleicacid polymer substrate, a tracer compatible with the restriction enzyme,and other components essential for the enzymatic reaction, enzymaticallycleaving the nucleic acid polymer substrate to form a restrictionproduct, analyzing the restriction product by an electrophoreticprotocol, and observing the tracer during the electrophoretic protocolwithout providing additional tracer beyond that which was included inthe reaction mixture.
 2. The method of claim 1, wherein the opticaldensity of the reaction mixture is at least about 15 at a visiblewavelength of maximal tracer absorbance.
 3. The method of claim 1,wherein the density of the reaction mixture is at least about 0.01 g/cmgreater than the liquid phase utilized in the chromatographic orelectrophoretic protocol.
 4. The method of claim 1, wherein the traceris amaranth dye.
 5. The method of claim 2, wherein the density of thereaction mixture is at least about 0.01 g/cm greater than the liquidphase utilized in the chromatographic or electrophoretic protocol, andthe tracer is amaranth dye.
 6. The method of claim 1 wherein the tracerin the reaction mixture is of adequate character and sufficient quantityto be visible during the electrophoretic protocol.